Loss of Microtubule Associated Protein 2 Immunoreactivity Linked to Dendritic Spine Loss in Schizophrenia

Microtubule-associated protein 2 (MAP2) is a neuronal protein that plays a role in maintaining dendritic structure through its interaction with microtubules. In schizophrenia (Sz), a number of studies have revealed that MAP2’s typically robust immunoreactivity (IR) is significantly reduced across several cortical regions. Previous studies have not explored the relationship between MAP2-IR reduction and lower dendritic spine density, which is frequently reported in schizophrenia nor has MAP2-IR loss been investigated in the primary auditory cortex (Brodmann Area 41), a site of conserved pathology in Sz. Last, the impact of chronic antipsychotic exposure is little understood. Using quantitative spinning disk confocal microscopy in two cohorts of Sz subjects and matched control subjects (Sz, n=20; C, n=20), we measured MAP2-IR as well as dendritic spine density and spine number in deep layer 3 of BA41. Sz subjects exhibited a significant reduction in MAP2-IR. The reductions in MAP2-IR were not associated with neuron loss, loss of MAP2 protein, clinical confounds, or technical factors; nor were MAP2-IR reductions linked with chronic haloperidol exposure in a macaque model. Dendritic spine density and number were also reduced in Sz and correlated with MAP2-IR. Twelve (60%) Sz subjects exhibited MAP2-IR values lower than the lowest controls; only in this group were spine density and number significantly reduced. These findings demonstrate that MAP2-IR loss is closely linked to dendritic spine pathology in Sz. Because MAP2 shares substantial sequence, regulatory, and functional homology with MAP tau, the wealth of knowledge regarding tau biology and the rapidly expanding field of tau therapeutics provide resources for identifying how MAP2 is altered in Sz and possible leads to novel therapeutics

Abnormal COX2/PGE2 Signalling in the Developing Cerebellum - A Link to Autism Spectrum Disorders

Autism spectrum disorders (ASDs) include a group of neurodevelopmental conditions that are characterized by deficits in social interaction and communication, increases in repetitive/restricted often stereotyped behaviour, and increases in anxiety. The heterogeneous nature of ASDs with regards to symptoms but also genetic profiles of ASD individuals, make understanding factors contributing to the disorder complex. However, literature suggests that ASDs arise from a combination of genetic and environmental factors. Clinical studies have suggested that abnormal lipid signalling, as a result of environmental insults can contribute to the etiology of ASDs. The phospholipid membrane of cells within the can be metabolized into lipid signalling molecules, including prostaglandins. Prostaglandin E2 (PGE2) is one of the most utilized lipid signalling molecules in the brain, involved in developmental processes such as synaptogenesis, migration, and differentiation of neuronal stem cells. Abnormal levels of PGE2, as well as COX-1 and COX-2, the rate-limiting enzymes in PGE2 synthesis have been linked to ASD. Furthermore, various environmental risk factors including exposure to heavy metals, infection/inflammation in pregnancy, exposure to pesticides, fragrances, and the use of over-the-counter medications such as aspirin and acetaminophen can affect PGE2 levels and are linked to ASD. The exact mechanisms that link abnormal COX2/PGE2 signalling to ASD are still unclear. To help address the lack of information, in this dissertation we first examine the effect of exposure to PGE2 on differentiated neuroectodermal (NE-4C) stem cells. Further, Studies have demonstrated that the cerebellum may be important in the etiology of ASDs. Interestingly there is evidence that PGE2 can affect postnatal development of the cerebellum. We examine the effect of increases (in a maternal PGE2 injection model) and decreases (in a COX-2--KI model) in PGE2 levels on prenatal neurodevelopment. We specifically examine the effects of these increases and decreases on cytoskeletal-dependent morphology through dendritic morphology within the cerebellum. Additionally, we examine the effect of prenatal PGE2-exposure on cerebellar-dependent motor function postnatally. Given the importance of sex as a factor in examining neurodevelopmental disorders such as ASD that have a large sex bias towards males, all of our in vivo studies address the modulation of the PGE2 effect by sex. These studies demonstrate that abnormal COX2/PGE2 signalling can affect important neurodevelopmental processes in vitro and development of the cerebellum in vivo. We observed disruptions in cytoskeletal dynamics, and changes in the expression of cytoskeletal proteins corresponding to abnormal COX2/PGE2 signalling. In PGE2-exposed mice, the changes in dendritic morphology in the cerebellum, corresponded to deficits in cerebellar motor function. Further, we found that the disruption of COX2/PGE2 affected development in a sex-dependent manner. The findings strengthen the involvement of COX2/PGE2 signalling in normal development of the brain and further suggest that abnormal COX2/PGE2 signalling as a result of exposure to environmental factors can result in neuropathologies including those found in ASD

REGULATION OF DENDRITIC SPINES BY 5-HT2A RECEPTOR SIGNALING PATHWAYS

Dendritic spines are small membranous protrusions from the dendrites of neuron, which are thought to serve as basic units of synaptic transmission, learning and memory. Disruptions in dendritic spine shape, size or number are associated with many brain diseases. Mounting evidence suggests that serotonin 2A (5-HT2A) receptors, the most abundant serotonin receptors in the prefrontal cortex, are involved in the regulation of dendritic spines. It has been suggested that both agonists (such as DOI) and antagonists (such as atypical antipsychotics) of 5-HT2A receptors can modulate different aspects of dendritic spines, however, the underlying mechanisms still remains unknown. In this dissertation, mechanisms underlying regulation of dendritic spines by both agonists and antagonists of 5-HT2A receptors are extensively studied and presented. I hypothesize that 5-HT2A receptor agonist regulate dendritic spines via transglutaminase- (TGase) catalyzed serotonylation of small G protein of the Rho family, whereas atypical antipsychotics change dendritic spines via activation of the Janus Kinase 2 (JAK2) signaling pathway. In the first study, the mechanisms and the functional consequences of 5-HT2A receptor-induced serotonylation of small G proteins of the Rho family were investigated in primary rat cortical neurons. Stimulation of 5-HT2A/2C receptors caused TGase-mediated transamidation and activation of Rac1 and Cdc42, but not RhoA, in both A1A1v cells and rat primary cortical culture. DOI-induced Rac1 transamidation occurs at Q61 in A1A1v cells, as demonstrated by site-directed mutagenesis at Q61 of Rac1. Furthermore, our findings were extended from 5-HT2A/2C receptors to another Gαq/11-coupled receptor, muscarinic acetylcholine receptors. In addition, stimulation of 5-HT2A/2C receptors by DOI leads to a transient dendritic spine enlargement, which was blocked by TGase inhibitor cystamine, suggesting 5-HT2A/2C receptors-induced transamidation of Rac1 and Cdc42 is involved in the regulation of dendritic spines by 5-HT2A/2C receptors. In the second study, to study the role of JAK2/ STAT pathway in the regulation of dendritic spines, Sprague-Dawley rats were pretreated with the JAK2 inhibitor AG490 or vehicle, followed by administration with olanzapine or vehicle daily for seven days. Microarray analysis of prefrontal cortices showed that 205 genes were significantly changed by AG490, olanzapine or the combination of both drugs compared to the controls. 92 of the 205 genes are changed by olanzapine via JAK2 signaling pathway. These genes are involved in the etiology of schizophrenia, neuronal signal transduction, neuronal growth factor, metabolism and energy, and synaptic plasticity. mRNA and protein levels of these genes were verified using real-time qPCR, western blot and the enzyme-linked immunosorbent assay (ELISA). Investigation on dendritic morphology shows that treatment with olanzapine induced a maturation in dendritic spines via both JAK2 dependent and independent pathways

Implementation of a chronic social stress rat model and the effects of allopregnanolone to reverse depressive and anxiety-like signs in nulliparous and primiparous females

Dissertação de mestrado em Ciências da SaúdeA depressão é uma doença psiquiátrica multidimensional, que afeta vários domínios do comportamento, e a mais prevalente a nível mundial. Além disso, é também a principal causa de incapacidade, sendo a exposição ao stress, nomeadamente stress social, um dos principais fatores desencadeadores do seu desenvolvimento. A maioria dos estudos pré-clínicos de avaliação da génese e tratamento na depressão são realizados em modelos animais de roedores do sexo masculino; no entanto, a depressão é duas vezes mais prevalente em mulheres do que em homens. A desregulação dos neuroesteróides, incluindo a alopregnanolona, está associada à depressão, nomeadamente em mulheres. A brexanolona, uma formulação aquosa e proprietária da alopregnanolona (Sage Therapeutics, Inc) foi recentemente aprovada pela FDA (Food and Drug Administration) como primeiro tratamento específico para a depressão pós-parto, abrindo a hipótese de que esta também possa ser utilizada para o tratamento de distúrbios depressivos especificamente em mulheres. Neste trabalho, implementou-se uma versão modificada de um protocolo de stress crónico de instabilidade social (CSIS) em ratos fêmea, com o objetivo de estudar a suscetibilidade de fêmeas nulíparas e primíparas para o desenvolvimento de sintomatologia depressiva. Implementaram-se 3 experiências distintas em fêmeas com idades e níveis de stress diferentes. Além disso, explorou se o potencial da alopregnanolona enquanto tratamento agudo para depressão induzida pelo stress, especificamente em fêmeas. O nosso estudo demonstrou que o protocolo de CSIS utilizado não induz comportamento do tipo depressivo ou anedonia em fêmeas em nenhuma das condições testadas, tendo, no entanto, produzido alterações fisiológicas descritas na depressão e comportamento do tipo ansioso, uma co-morbilidade importante na depressão. Verificou-se ainda que a idade, intensidade do stress e experiência reprodutiva afeta diferencialmente a resposta das fêmeas nulíparas e primíparas a este protocolo de stress. Tendo em consideração as diferenças fenotípicas encontradas, este estudo demonstra a necessidade de aprofundar o estudo dos mecanismos fisiopatológicos específicos da depressão em fêmeas.Depression is a multidimensional psychiatric disorder, affecting several behavioral domains, and the most prevalent psychiatric disorder worldwide. Moreover, it is the leading cause of disability, with exposure to stress, namely social stress, being one of the main triggers for its development. Whereas depression is reportedly twice more prevalent in women than in men, most preclinical studies are performed in male rodents. Of notice, females’ physiology and reproductive experience are associated with changes to endocrine profiles, brain and behavior that may influence both stress and treatment response, supporting the need for animal models of depression considering these factors. Dysregulation of neuroactive steroids, including allopregnanolone, is associated with depression. Interestingly, a proprietary formulation of allopregnanolone – brexanolone (Sage Therapeutics, Inc.) - has been recently approved specifically for the treatment of postpartum depression, opening the hypothesis of whether it could also be used for treating depression, specifically in females. In this study, we have implemented a modified version of a chronic social instability stress (CSIS) protocol in female rats, to assess the susceptibility of nulliparous and primiparous female rats for the development of depressive-like signs. Moreover, we have explored the potential use of allopregnanolone for the acute treatment of stress-induced depressive-like behavior specifically in females. The CSIS protocol used did not induce depressive-like behavior or anhedonia in females under any of the conditions tested, but produced anxiety-like behavior, an important co-morbidity in depression, and physiological changes described in depression. Molecular and cellular parameters were also assessed revealing that exposure to social stress affects neuronal morphology, gene expression and neuronal connectivity, specifically in primiparous females. Given the phenotypic differences we found linked with age, stress intensity and reproductive experience and the little information concerning female animal models, this work emphasizes the need for further studying the specific pathophysiological mechanisms of depression in females.Este projeto foi apoiado por bolsas da Plataforma de Microscopia Científica do ICVS, membro da infra-estrutura nacional PPBI - Plataforma Portuguesa de Bioimagem (PPBI-POCI-01-0145-FEDER-022122; por fundos nacionais, através da Fundação para a Ciência e Tecnologia (FCT) - projeto UIDB/50026/2020 e IF/01079/2014; e pelos projetos NORTE-01-0145-FEDER-000013 e NORTE-01-0145-FEDER-000023; pelo Programa Operacional Regional Norte Portugal (NORTE 2020), no âmbito do Acordo de Parceria PORTUGAL 2020, através do Fundo Europeu de Desenvolvimento Regional (FEDER), pela Fundação BIAL (Bolsa 427/14), e pelo prémio Nature Research Award for Driving Global Impact. Este trabalho contou ainda com o apoio financeiro da Bn'ML – Behavioral and Molecular Lab

ENDOCANNABINOID REGULATION OF ADOLESCENT DEVELOPMENT IN MALE AND FEMALE RATS

The present thesis investigated the contributions of adolescent endocannabinoid signalling to brain and behaviour development in male and female rats. In chapter 2, daily administration of the CB1 antagonist AM251, alone or in tandem with a psychological stressor, increased social interactions, reduced dorsal hippocampal CB1 expression, and increased mPFC GAD67 expression in female rats 24-48 h after treatment, with no effects in males. In chapter 3, adolescent CB1 antagonism reduced anxiety in adult males, with no effects in females. Conversely, adolescent AM251 increased contextual fear in adult females, with no effects in males. In chapter 4, AM251 females spent more time initiating social interactions after a 5-day drug washout period than vehicle females, with no effects in males. To identify brain regions underlying the effects of AM251 on social behaviours, I repeated social interaction testing in vehicle and AM251 females and collected brains for immunohistochemical labelling of EGR-1 as a marker of neural activation in the CA1, CA2, and CA3 subfields of the dorsal hippocampus and the shell and core divisions of the nucleus accumbens (NAc). Consistent with my previous findings, AM251 females spent more time initiating social interactions and had greater EGR-1 cell counts in the NAc shell than vehicle females, with no group differences in the NAc core or in any of the hippocampal subfields investigated. EGR-1 cell counts in the dCA2 were negatively correlated with social interactions in vehicle and AM251 females. A positive correlation between NAc shell EGR-1 cell counts and social interactions was observed only in AM251 females. Regression analysis using drug treatment and EGR-1 cell counts in dCA2 and NAc shell resulted in a model with an adjusted R2 of 0.90. Both drug treatment and EGR-1 cell counts in the dorsal CA2 emerged as unique predictors of individual differences in social interaction, and drug and NAc shell EGR-1 cell counts interacted to significantly predict social interactions in AM251 females only. Together, these studies provide support for sex-specific contributions of endocannabinoid signalling to the development of brain and behaviour in adolescence in male and female rats

Physiological Effects of Estradiol in the Mouse Hippocampal Formation

At several points in a woman’s life, changes in circulating estradiol are associated with disturbances in mood and cognitive function. To determine the biological basis of these behavioral changes, researchers have concentrated on the hippocampal formation, a medial temporal lobe structure involved in the regulation of mood and cognition in humans. It is now clear that estradiol increases the substrates of hippocampal synaptic plasticity, including dendritic spine density, synapse density, and synaptic protein expression. In some cases, these changes are associated with alterations in mood and hippocampal-dependent learning and memory. The upstream mediators of these estradiol effects remain unknown, but likely candidates may be inferred from known regulators of hippocampal synaptic plasticity and estradiol effects in other tissues. This thesis explored estradiol activation of two of these candidates, PI3 kinase/Akt and brain-derived neurotrophic factor (BDNF), in the dorsal hippocampus of female mice. In naturally cycling and ovariectomized female mice, estradiol increased hippocampal Akt and BDNF signaling as determined by immunocytochemistry and in situ hybridization. These effects were associated with changes in synaptic protein expression, and required specific estrogen receptor isoforms, as demonstrated using estrogen receptor knockout mice. Immuno-electron microscopy revealed that estradiol specifically increased signaling through presynaptic TrkB receptors in Schaffer collateral and mossy fiber axons. Estradiol effects on Akt signaling were associated with changes in spatial memory ability, and were profoundly altered in mice expressing the BDNF variant Val66Met. This study therefore identifies Akt and BDNF signaling as important upstream pathways in the control of hippocampal function by ovarian steroids in vivo. Circulating estradiol activates Akt rapidly through a BDNF-dependent mechanism, and increases expression of BDNF more slowly, leading to increased activation of the BDNF receptor TrkB. After activation, Akt and TrkB may participate in estradiol regulation of spine synapse density and hippocampal cell excitability. Future studies should aim to understand the specific role of Akt and BDNF in estradiol modulation of hippocampal function and behavior

Translational assessment of cognitive impairments in depression models

Major depressive disorder (MDD) affects 300 million people worldwide and is a major contributor to the global burden of disease. The aetiology of depression, emerging through a gene x environment interaction, is still incompletely understood which prevents tailoring of treatment approaches. In addition to MDD core symptoms, such as anhedonia (a diminished anticipation or experience of pleasure), depressed patients suffer from a plethora of manifestations including cognitive impairments, which occur primarily in the domains of executive function, attention and memory. Patients remitted from affective symptoms of MDD often continue to display cognitive impairments. These cognitive deficits are the longest present residual symptom, predict treatment response and increase risk of relapse. Consequently, cognitive impairments need to be targeted more effectively by antidepressants for complete remission from MDD. Clinically relevant animal models are essential for developing, tailoring and testing such novel, pro-cognitive antidepressants. This PhD project aimed to establish a preclinical screening platform for the testing of pro-cognitive antidepressants, to improve understanding of MDD risk factors and consequent symptom development, and finally, to focus on clinical relevance of the applied techniques. The chronic mild stress (CMS) rodent model of depression was used, known for displaying the core symptom anhedonia, but also for a high construct, face and predictive validity. The environmental MDD risk factor ‘stress’ induces an anhedonic-like phenotype in a subgroup of exposed rats, whereas another subgroup of rats is resilient, as determined by the sucrose consumption test. The cognitive performance of different rat strains, including CMS anhedonic-like and resilient rats, was assessed employing the touchscreen operant platform, which was developed based on the Cambridge neuropsychological test automated battery (CANTAB) for assessing cognition in humans. Furthermore, a group of anhedonic-like rats was treated with the antidepressant vortioxetine, which acts as both a pro-cognitive and antidepressant treatment. Our results showed that stress exposure induced anhedonia in albino and pigmented rat strains, although stress did not affect cognitive performance of pigmented rats in a simple pairwise discrimination touchscreen task. Applying a more complex pairedassociates learning touchscreen task revealed impaired cognitive performance in the CMS anhedonic-like but not in the resilient phenotype. Furthermore, vortioxetine treatment reversed anhedonia in the CMS model and altered executive functions in treated rats. The expression of genes involved in the stress response, affective disorders and neuronal plasticity was altered in the prefrontal cortex and hippocampus owned to treatment and hedonic state. Thus, we have demonstrated that the CMS model exhibits both stress-induced cognitive alterations and depression-associated cognitive impairments in touchscreen tasks. Furthermore, touchscreen testing was sufficiently sensitive to detect alterations in cognitive performance due to pharmacological intervention. Overall, we established a potential platform for pro-cognitive antidepressant drug screening. Furthermore, brain derived neurotrophic factor (BDNF), involved in learning and memory, was examined in the context of depression. BDNF is reduced in MDD patients as well as in preclinical models in response to stress. Although this suggests that BDNF contributes to the aetiology of depression, studies including mice heterozygous for BDNF (BDNF+/-) have generated conflicting results. BDNF+/- rats may provide a more suitable model as (1) rats have a greater behavioural repertoire than mice, (2) classical behaviour tests are designed for rats, and (3) rats, like humans, produce peripheral BDNF. We found anhedonia and mild signs of anxiety in BDNF+/- rats, accompanied by prefrontal and hippocampal changes in expression of genes relevant in psychiatric disorders and underpinning learning. Thus, behavioural and molecular findings in BDNF+/- rats complement existing literature and suggest that rats are a more suitable model in BDNF research than mice. Overall, the project uncovered environmental and genetic manifestations of risk factors in translational models and established a novel tool for translational pro-cognitive antidepressant drug screening